The classic model of blood coagulation proposes a series of cascading reactions that result in clot formation. This model has been modified to incorporate initiation, amplification, and propagation phases. Furie and Furie, (1992) N. Engl. J. Med. 326:800; Roberts and Lozier, (1992) Hosp. Pract. 27:97. Adding an additional layer of complexity is an array of mechanisms that turn off and/or modulate the coagulation cascade. Thrombin is a key regulatory point in pathways that lead to blood coagulation, as well as those that reverse this process. Circulating prothrombin is cleaved to form thrombin following interaction with other blood clotting factors. Thrombin is a trypsin-like serine protease, which is responsible for the proteolytic conversion of fibrinogen to fibrin, with the aggregation of fibrin molecules resulting in the formation of a "soft" clot.
The proteolytic actions of thrombin are inhibited by interaction with plasma serine proteinase inhibitors ("serpins"), such as heparin cofactor II (HCII), antithrombin III (ATIII), protein C inhibitor (PCI), and .alpha.-proteinase inhibitor. Thrombin recognizes a conserved amino acid sequence among serpins, termed the "reactive site loop", as a potential substrate. Interaction of thrombin with the reactive site loop results in the formation of a thrombin-serpin complex that is essentially irreversible. Complex formation inactivates both the thrombin protease and serpin inhibitory activities. Pratt and Church, (1993) Blood Coag. and Fibrinol. 4:479.
The inhibitory actions of ATIII, PCI and HCII are accelerated in the presence of heparin (1000-10,000 fold), and the anti-coagulation effects of heparin are believed to be mediated through these plasma serpins. HCII is the most dependent of these inhibitors on glycosaminoglycan; it has only about 10% the inhibitory activity of ATIII or PCI in the absence of heparin. Pratt et al., (1989a) Thromb. Res. 53:595; Pratt et al., (1989) Ann. N.Y. Acad. Sci. 556:104; Pratt et al., (1992) J. Biol. Chem. 267:8795; Pratt and Church, (1992) J. Biol. Chem. 267:8789. Thrombin inhibition by HCII, but not ATIII, is also accelerated in the presence of dermatan sulfate and dermatan sulfate proteoglycans. Pratt and Church, (1993) Blood Coag. Fibrinol. 4:479. Moreover, ATIII inhibits most of the serine proteases involved in coagulation, whereas HCII appears to be uniquely specific for thrombin. Church and Hoffman, (1994) TCM 4:140. The only other known substrates for HCII are chymotrypsin, cathepsin G, and Streptomyces gresius Protease B. Church et al., (1985) Proc. Natl. Acad. Sci. USA 82:6431; Parker and Tollefsen, (1985) J. Biol. Chem. 260:3501; Pratt et al., (1989b) Ann. N.Y. Acad. Sci. 556:104.
As described above, heparin is a known anti-coagulant, and it is widely-administered to human and animal subjects for this purpose. Individual response to heparin is quite variable, however, and heparin administration can result in hemorrhaging, thrombocytopenia, and osteoporosis. Stone, (1995) TCM 5:134; U.S. Pat. Nos. 5,118,793 and 5,102,995 to Tollefsen et al. Accordingly, there exists a need in the art for alternative anti-coagulant agents and therapies.